Approach 1. A region of the zebrafish embryos yolk, the yolk sycytial layer (YSL) contains essential signals for mesoderm and endoderm induction. This year we analyzed ChIP-seq data revealing the binding site of the central TSL transcription factor we have been studying: Mxtx2. We have further generated several reports constructs for testing requirements endogenous iterations of this binding site in Mxtx2 target genes of interest. Approach 2. Modeling complex genetics underlying human disease in zebrafish. We have established a new zebrafish mutant genetic line in a Nodal pathway antagonist and have used this line in a series of studies to establish that simultaneously carrying Nodal and Nodal antagonist mutations confers protection against the acquistion of cyclopia, analogous to human holoprosencephaly, but increases the risk of laterality defects. A manuscript is being prepared. Approach 3. Identifying roles of RhoGTPase signaling in gastrulation movements. Background: RhoGEF proteins are positive regulators of RhoGTPases, which have profound roles in cellular movement and morphology. To identify RhoGEFs with roles in directing the morphogenetic events of gastrulation, we performed a loss-of-function screen. We identified 48 RhoGEFs expressed during early embryogenesis and determined the loss-of-function phenotypes for 23 of these, using a non-invasive embryo holding system we designed that allows for the parallel time-lapse documention of 54 embryos. We thus identified five RhoGEFs for which two independent MOs produced the same phenotype. Three of these, homologues of ARHGEF16, Frabin and Net1, respectively, disrupted epiboly. Two others, ARHGEF10 and PLEKHG4 homologues, caused post-gastrulation defects during somitogenesis stages. We have demonstrated the specificity of the Frabin and Net1 MOs by performing mRNA rescue experiments. A manuscript was submitted and received favorable reviews. Approach 4. Defining roles and interactions of transcription factors in the early mesoderm and endoderm. We previously developed a novel approach to identify many of the genes expressed in nascent mesoderm and endoderm. Last year we have embarked in a revisitation of this project, but using deep sequencing to get a much more exhaustive list of expressed genes. We completed the deep sequence analysis and generated a comprehensive list of transcription factors expressed during this critical stage of interest. We ordered Nanostring-based code sets that will be used in the next fiscal year to define the temporal expression of these transcription factors.